In our prior U.S. patent application Ser. No. 11/735,869, filed Apr. 16, 2007, we described a treatment for patients suffering from chronic myocardial infarction by injecting autologous bone marrow cells in or near the areas of chronic myocardial infarction in the patient's heart. In addition to the mononuclear cells, mesenchymal cells, CD-34 positive cells, CD-90 positive cells, and CD-133 positive cells mentioned in therein, additional cell therapies may be applicable to other cardiac conditions. Each of these cell therapies may be useful to treat chronic myocardial infarction, acute myocardial infarction, myocardial ischemia, chronic myocardial ischemia, heart failure, cardiovascular disease, and peripheral vascular disease. U.S. patent application Ser. No. 11/735,869, U.S. is hereby incorporated by reference.
Human bone marrow mesenchymal cells (hMSCs) are currently being investigated for a number of clinical applications including cardiovascular repair, orthopaedic repair, connective tissue repair, and immune diseases such as graft versus host disease, Crohn's disease. They have potential roles in other immune diseases such as lupus, osteoarthritis and rheumatoid arthritis as well as well as diabetes. They also have potential to act as carriers of gene-based therapeutics with ex vivo transfection of the cells to enhance the control of gene based therapeutics dosing. The following prior art references are hereby incorporated by reference: U.S. Pat. Nos. 7,101,704; 7,029,666; 6,875,430; 6,863,900; 6,835,377; 6,797,269; 6,761,887; 6,709,864; 6,685,936; 6,541,024; 6,387,369; 6,387,367; 6,379,953; 6,368,636; 6,358,702; 6,355,239; 6,342,370; 6,328,960; 6,322,784; 6,281,012; 6,261,549.
Although it has been argued that allogenic cells have great potential to provide an off the shelf product for patients because they will not be recognized as foreign by the recipient patient, this has not yet been proven. Allogenic cells have a greater propensity (compared to autologous cells) to carry diseases from the donor to the recipient and have a greater propensity for rejection of the therapeutic cells by the immune system of the patient who received them.
Expansion of autologous hMSC in a truly autologous culture medium that contains no animal serum or allogenic culture additives for clinical transplantation to the patient, which has not previously been proposed, should also be valuable avoiding the problems associated with allogenic cells. To date, reported clinical trials are employing human bone marrow mesenchymal stromal cells generated in a culture medium supplemented with fetal calf serum (FCS). FCS is an undesired source of xenogeneic antigens and bears the risk of transmitting animal viral prion and zoonose contaminations. Additionally FCS has been implicated with anaphylactic or arthus like immune reactions in patients who received cells generated in FCS supplemented medium even leading to arrhythmias after cardioplasty (Chachques 2004).
Early work on autologous serum hMSC has recently been shown to be promising by Stute et al 2004, but the amount of autologous serum required is identified as a significant hurdle to clinical expansion such that the investigators proposed using 1% and 3% autologous serum in the culture medium even though they had far superior results at 10% autologous serum. Other investigators have also shown a faster proliferation compared to FCS at least during first passages without loss of the typical phenotype, motility, and differentiation capabilities in vitro, whereas allogenic human serum resulted in hMSC growth arrest and death (Kobayashi et al 2005, Shandahar et al 2005, Mizuno et al 2006).
The primary reason presented that autologous serum cannot be considered as a general substitute for FCS is the amount of autologous serum necessary for sufficient expansion would exceed the amount a donor could provide (Lange et al 2007). Lange et al 2007 proposed the use of pooled platelet lysates to create an activated plasma to avoid the use of FCS, but their solution falls short of that provided here in that they are pooling the platelets from multiple donors to create an allogenic culture medium.
Here we disclose three methods to culture bone marrow or adipose tissue derived autologous culture medium expanded autologous mesenchymal stem cells (ACMEAM's) from the bone marrow or adipose tissue. These inventive methods have enormous value and have been validated in both a swine and human and are comparable to the gold standard of culturing in 10% fetal calf serum.